T lymphocytes recognize a variety of foreign antigens through the cell-surface glycoproteins known as T cell receptors (TCRs). Recognition of peptide antigens presented by major histocompatibility complex (MHC) molecules is mediated by diverse (xp heterodimers of the TCR. The Voc and Vp domains of TCRs form the recognition surface for MHC/peptide complexes. In order to facilitate NMR studies of the V(xNp heterodimer, the Voc and Vp domains were expressed as a single polypeptide in E. coli. The solution structure of a single-chain T cell receptor (scTCR) derived from the MHC class II-restricted DIO TCR was determined using NMR spectroscopy. The scTCR is a 27.8 kD protein comprising 255 amino acids. The structure was determined using heteronuclear, multi-dimensional NMR spectroscopy using protein enriched in "N, "C and 2 H isotopes. The conformations of complementarity determining regions (CDRs) 3P and I a and surface properties of 2a are different from related class I-restricted TCRs. A conserved orientation for TCR Vadomains in complexes with both class I and II MHC/peptide ligands is inferred, implying that small structural variations in V(X confer MHC class preference. High mobility of CDR3 residues relative to other CDR or framework residues (picosecond timescale) provides insight into immune recognition and selection mechanisms. I